The long-term goal of this research program is an understanding of how clathrin coated membranes contribute to intracellular organization and membrane dynamics. Our approach to these issues has been to study the structure, activities and interrelationships of the various components of the clathrin coated membrane at the molecular level. Clathrin, the major protein component of the coated membrane, interacts with AP-1 or AP-2, which have been termed assembly, adaptor or associated proteins, to form the completed lattice structure that is found within many different cell types. Another AP-like protein, AP-3, is a major neuronal protein which is highly conserved but little is known about its interactions with clathrin and there are no compelling hypotheses for its intracellular functions. Detailed information will be obtained on the in vitro interaction of AP-3 with clathrin. Using laser confocal immunofluorescence microscopy, the in situ localization of AP-3 in rat sympathetic neurons in culture will be determined. These studies will provide needed criteria for evaluating possible AP-3 functions. During the last grant period, studies in this laboratory on cultured neurons and optic nerve have identified a soluble complex of clathrin in stoichiometric association with hsc7O/uncoating protein and a novel 100K protein. The majority of cellular clathrin was found in another complex, also containing hsc7O but lacking the 100K protein. The latter complex resists extraction and has been operationally termed "cytoskeletal" clathrin. Confocal microscopy and conventional immunofluorescence will be used to determine the intracellular localization of these complexes in intact and permeabilized neurons and on isolated membrane surfaces. The biochemical composition of these complexes, their interrelationship and the identity of the 100K protein will be determined. The hypothesis that "soluble" and "cytoskeletal" clathrin reflect disassembled clathrin intermediates and assembled coated membrane, respectively, will be evaluated. This information is required for an accurate understanding of clathrin dynamics and its function in coated membrane-mediated events such as receptor-mediated endocytosis and Golgi processing. These processes are critical to the existence of all higher eukaryotic cells.